The Paramyxovirus SV5 Small Hydrophobic (SH) Protein Is Not Essential for Virus Growth inTissue Culture Cells

Biao He,* George P. Leser,† Reay G. Paterson,† and Robert A. Lamb*,†,1

*Howard Hughes Medical Institute and †Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston,Illinois 60208–3500

Received June 26, 1998; returned to author for revision July 15, 1998; accepted July 29, 1998

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) andhemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein(SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm.To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA cloneof the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5(rSV5DSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis ofproperties of rSV5DSH indicated that lack of expression of SH protein did not alter the expression level of the other virusproteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a newcontent mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 andrSV5DSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting,examination of purified rSV5DSH by electron microscopy did not show an altered morphology from SV5. Thus in tissueculture cells the lack of the SV5 SH protein does not confer a recognizable phenotype. © 1998 Academic Press

INTRODUCTION

Simian virus 5 (SV5) is a member of the Rubulavirusgenus of the family Paramyxoviridae, a genus which in-cludes mumps virus, human parainfluenza virus type 2(HPIV2) and type 4 (HPIV4), and Newcastle disease virus(NDV). Other members of the Paramyxoviridae include Sen-dai virus, human parainfluenza virus type 3, measles virus,canine distemper virus, rinderpest virus, and respiratorysyncytial (RS) virus. SV5 contains a negative-sense single-stranded RNA of 15,246 nucleotides and encodes eightknown viral proteins: nucleocapsid protein (NP), V proteinand phosphoprotein (P), matrix protein (M), fusion protein(F), small hydrophobic integral membrane protein (SH),hemagglutinin-neuraminidase (HN), and polymerase pro-tein (L). The P protein mRNA is synthesized through acotranscriptional RNA editing process in which two non-templated G residues are inserted into the templatedmRNA transcript (Thomas et al., 1988). The NP, P, V, and Lproteins are associated with the RNA genome to form thenucleocapsid core; these proteins form the viral transcrip-tion/replication complex. The M protein is a peripheralmembrane protein, and the SH, F, and HN proteins areintegral membrane proteins. F and HN are involved in viral

entry into cells and HN is important for virus release fromcells (reviewed in Lamb and Kolakofsky, 1996).

The SV5 SH gene is located between the genes for Fand HN (Hiebert et al., 1985a,b). The SH protein contains44 amino-acid residues, and in SV5-infected cells SHprotein is orientated in membranes with its N terminus inthe cytoplasm. SH protein contains a transmembranedomain with a predicted length of 19 residues and anectodomain of 7 residues (Hiebert et al., 1985b, 1988).The function of the SH protein in the SV5 life cycle is notknown, and it has not been determined previously if SHis a component of virions.

The SH gene is not common to all members of theParamyxoviridae, and the only other virus that contains ananalogous gene located between the genes for F and HNis the closely related Rubulavirus, mumps virus (Elango etal., 1989; Takeuchi et al., 1996). In the Pneumovirus, respi-ratory syncytial (RS) virus, a gene encoding a third integralmembrane protein, designated SH, has been identified(Collins and Wertz, 1985; Olmsted and Collins, 1989). How-ever, the RS virus SH protein is considerably larger thanthat of SV5, and it is not known if the RS virus SH protein isa functional counterpart of the SH protein. The RS virus SHgene is lacking from an attenuated strain of RS virus (Karronet al., 1997) and a recombinant RS virus lacking the SHgene is viable in tissue culture (Bukreyev et al., 1997).

Recently, a reverse genetics system was developed forSV5 such that infectious virus (rSV5) could be rescued fromcDNA (He et al., 1997): the scheme used was first de-

1 To whom correspondence and reprint requests should be ad-dressed at Department of Biochemistry, Molecular Biology and CellBiology, Northwestern University, 2153 North Campus Drive, Evanston,IL 60208-3500. Fax: (847) 291-2467. E-mail: ralamb@nwu.edu.

VIROLOGY 250, 30–40 (1998)ARTICLE NO. VY989354

0042-6822/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

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scribed for rabies virus (Schnell et al., 1994) and subse-quently has been extended to several members of theMononegavirale (reviewed in Roberts and Rose, 1998). Tostudy properties of the SH protein in the life cycle of SV5,we have deleted the SH gene from the infectious cDNA andhave shown that even though SH is a component of virions,a viable virus (rSV5DSH) can be rescued in tissue culturethat lacks expression of the SH protein: we describe prop-erties of rSV5DSH.

RESULTS

Generation of rSV5DSH

The plasmid pSV5DSH was derived from the infectiouscDNA of SV5 (pSV5). The SH gene and the gene end (GE)and gene start (GS) sequences between SH and HN weredeleted. The final genome length of pSV5DSH (15,012 nu-cleotides) was designed such that the genome length wasa whole integer divisible by six to adhere to the ‘‘rule of six’’(Calain and Roux, 1993). It was anticipated that rSV5DSHtranscription of the HN gene would use the SH gene startsequences and thus the HN mRNA 59 nontranslated regionwould be extended in length over that of rSV5. To recoverviruses, plasmids pSV5 and pSV5DSH were transfected,together with the helper plasmids encoding NP, P, and L

proteins, into A 549 cells, which had been preinfected witha modified vaccinia virus encoding a T7 RNA polymerasegene (MVA). Recovery of viruses was performed as de-scribed previously (He et al., 1997).

Recovery of rSV5DSH was indicated initially by the ob-servation of syncytia in CV-1 cells after infection with thesupernatant from the transfected A549 cells. To confirm thatrSV5DSH was recovered, total RNAs were extracted fromCV-1 cells, and the RNAs subjected to RT–PCR using ap-propriate primers as indicated in Fig. 1. Cells transfectedwith pSV5 yielded a DNA fragment (576 bp) that was foundto be larger than the product from cells transfected withpSV5DSH (342 bp) (Fig. 1, lanes 6 and 7). Appropriatecontrols for the RT–PCR reaction were negative (Fig. 1,lanes 1–5). The RT–PCR products from rSV5- and rSV5DSH-infected cells were purified and their nucleotide sequenceobtained. The resulting sequences matched the pSV5 andpSV5DSH plasmid sequences, confirming that rSV5 andrSV5DSH had been recovered.

rSV5DSH mRNA synthesis

To examine transcription of the genome of SV5 andrSV5DSH between the F and HN genes, the accumula-tion of the F, SH, and HN mRNA in infected CV-1 cells

FIG. 1. Recovery of rSV5DSH. Top: Structure of the rSV5 and rSV5DSH genomes. The SH coding region and the GE/GS sequences between SHand HN, which control termination, polyadenylation, and reinitiation of SV5 transcription, were deleted from an infectious cDNA of the SV5 genome.As a result, transcription of the HN gene uses the GS sequence from the SH gene. Rescue of rSV5DSH was performed as described under Materialsand Methods. Bottom: RT–PCR of RNA from rSV5 and rSV5DSH virus-infected CV-1 cells using primers BH191 and BH194. The products were analyzedon 1% agarose gels: lane 1, PCR control, which contained no template; lanes 2, 4, and 6, products from rSV5-infected cell; lanes 3, 5, and 7, productsfrom rSV5DSH-infected cells; lanes 2 and 3, RT control in which no RT was added in the RT reaction; lanes 4 and 5, PCR control in which no Taq DNApolymerase was added; lanes 6 and 7, complete RT-PCR reaction; lane 8, DNA size marker with relevant sizes indicated.

31DELETION OF PARAMYXOVIRUS SV5 SH GENE

was examined by hybridization of purified poly(A)-con-taining RNA, using a Northern blot analysis. As shown inFig. 2, hybridization with a probe specific for SH indi-

cated that the SH-specific mRNA was lacking fromrSV5DSH-infected cells, together with the F–SH andSH–HN RNA readthrough products observed with SV5(Fig. 2 and see Paterson et al., 1984). However, accumu-lation of the F and HN mRNAs between SV5- andrSV5DSH-infected cells appeared to be comparable. Aslightly slower mobility of the rSV5DSH HN mRNA wasdiscernible compatible with the HN mRNA of rSV5DSHcontaining a longer 59 noncoding region than SV5 due toconstruction of pSV5DSH.

rSV5DSH protein synthesis

To examine viral-specific protein accumulation, CV-1cells were infected with SV5, rSV5, and rSV5DSH and met-abolically labeled with [35S]-Promix, cells lysed and pro-

FIG. 3. Protein expression in rSV5- and rSV5DSH-infected cells.Mock-infected and SV5-, rSV5-, and rSV5DSH-infected CV-1 cells weremetabolically labeled with 100 mCi/ml of [35S]-Promix for 2 h at 22 h p.i.and cell lysates were prepared for immunoprecipitation reactions. (A)Immunoprecipitation using a rabbit sera prepared against disruptedSV5 virions. (B) Immunoprecipitation using sera specific for HN, SH, F2,P, and V (see Materials and Methods).

FIG. 4. (Top) Immunofluorescent staining patterns of rSV5- and rSV5DSH-infected cells. CV-1 cells on coverslips were mock infected or infectedwith rSV5 or rSV5DSH and at 24 h p.i. were fixed with 1% formaldehyde and permeabilized with 0.1% saponin. Cells were stained with mouse mAbsspecific for F and HN and rabbit polyclonal sera specific for SH and FITC-conjugated goat anti-mouse or Texas-Red-conjugated goat anti-rabbitsecondary antibody. Fluorescence was examined using a Nikon FXA fluorescent microscope and appropriate filter sets.

FIG. 5. (Middle and bottom) Fusion activity of rSV5- and rSV5DSH-infected cells. (A) Lipid mixing assay. Octadecyl rhodamine (R18)-labeled RBCswere overlaid on SV5-infected CV-1 cells at 4°C. The temperature was shifted to 37°C and after 0, 5, and 15 min the transfer of R18 to the CV-1 cellswas examined by confocal microscopy. (B) Content mixing assay. One set of BHK-21F cells in six-well plates were infected with rSV5 or rSV5DSH for1 h and then transfected with plasmid pYZ3, which encodes bacteriophage T7 RNA polymerase under the control of the SV40 late region promoter.A second set of BHK-21F cells on 6-cm-diameter plates was transfected with pBH82, which encodes the CAT gene under the control of a T7 RNApolymerase promoter. After 10 h incubation the pBH82-transfected cells were trypsinized and overlaid on the pYZ3-transfected cells. At 4, 8, and 12 hafter mixing the cells were harvested and CAT protein activity was measured as described under Materials and Method. CAT activity is in arbitraryunits; N 5 3.

FIG. 2. Northern blot of SV5- and rSV5DSH-infected cell RNA. Poly(A)-containing RNA species from SV5- and rSV5DSH-infected cells wereisolated on oligo-(dT) cellulose and separated by electrophoresis on a1.5% agarose/2.2 M formaldehyde gel and transferred to a nitrocellu-lose membrane as described under Material and Methods. Left: 32P-labeled cDNA probes specific for both the F and SH genes werehybridized to the filter. Right: 32P-labeled probes specific for HN and SHwere hybridized to the filter. The mRNAs for HN, F, and SH are shownand the identity of the readthrough transcripts, M–F, F–SH, and SH–F isbased on their expected mobilities as compared to size markers (notshown). A slightly slower mobility of HN mRNA from rSV5DSH-infectedcells is a result of the altered GS sequence (see Fig. 1).

32 HE ET AL.

teins immunoprecipitated with specific antisera. As shownin Fig. 3A, when a serum generated to disrupted SV5 virionswas used, the overall polypeptide pattern observed amongviruses was indistinguishable. The use of specific sera forHN, F2, P, and V indicated similar levels of accumulation ofthese proteins, and the use of SH-specific sera indicatedthat whereas SH protein could be identified in SV5- andrSV5-infected cells, it could not be identified in rSV5DSH-infected cells (Fig. 3B).

Deletion of the SH gene does not affect thesubcellular localization of F and HN or affect virus-mediated fusion

The SV5 F protein only requires HN for cell attachmentpurposes, unlike many other paramyxoviruses, whichrequire the action of their homotypic HN protein for afusion reaction to proceed (reviewed in Lamb, 1993).However, a possible role of the SH protein in participat-ing in the fusion reaction had not been tested rigorously.For RS virus, the SH protein had been shown to berequired for the fusion reaction using in vitro assays(Heminway et al., 1994); however, a viable RS virus lack-ing the SH gene has been recovered (Bukreyev et al.,1997). To test for a possible effect of the SV5 SH proteinon the fusion reaction, the intracellular staining patternsof SH, F, and HN were examined. Immunofluorescentstaining of F, SH, and HN proteins in rSV5 and rSV5DSHvirus-infected cells were all comparable, indicating thatSH protein expression did not cause a subcellular relo-calization of the F and HN proteins (Fig. 4). Furthermore,analysis of the amounts of F and HN at the surface ofrSV5- and rSV5DSH-infected cells by flow cytometry in-dicated comparable surface expression levels of the twoglycoproteins (Table 1).

To examine an affect of deletion of the SH gene from

SV5 on fusion, both lipid and content mixing assays wereperformed. In the lipid mixing assay, the fluorescent dyeoctadecyl rhodamine (R18), contained in human erythro-cyte membranes, was transferred to the membranes ofCV-1 cells infected with rSV5 or rSV5DSH. Observation ofthe time course of dye transfer to the CV-1 cells, byconfocal microscopy, indicated a lack of a discernibledifference between rSV5- and rSV5DSH-infected cells(Fig. 5A).

To examine mixing of the aqueous contents of thecytoplasm of two cells upon fusion, we developed a newassay. The established assay involving transfection of aplasmid encoding b-galactosidase, under the control of abacteriophage T7 RNA polymerase promoter, into onecell and expression of T7 RNA polymerase (from a re-combinant vaccinia virus) in another cell has proven veryuseful when fusion proteins are expressed from cDNAs(Nussbaum et al., 1994; Bagai and Lamb, 1995–1997).However, we were concerned about the cytopathic effectof vaccinia virus coinfection on SV5-infected cells. Thuswe modified the established assay such that BHK-21Fcells were infected with rSV5 or rSV5DSH and thentransfected with pYZ3, a plasmid that encodes a gene forT7 RNA polymerase under the control of the SV40 lateregion promoter and polyadenylation signal. A secondbatch of BHK-21F cells were transfected concomitantlywith pBH82, a plasmid encoding chloramphenicol acetyl-transferase (CAT) under the control of a T7 RNA poly-merase promoter and an internal ribosome entry site(EMCV–IRES). At 8–10 h posttransfection, the pBH82-transfected cells were trypsinized and overlaid onto thepYZ3-transfected/SV5-infected cells At 4, 8, and 12 hpostmixing of the cells, CAT activity was measured. Asshown in Fig. 5B, CAT activity, which is indicative ofcytoplasmic content mixing, was very similar for rSV5and rSV5DSH virus-infected cells.

SH protein is a component of purified SV5 virions

As SH is an integral membrane protein expressed atthe surface of virus-infected cells (Hiebert et al., 1988), itseemed likely that SH would be incorporated into bud-ding particles, unless a mechanism exists for excludingits presence from virions. To determine whether SH pro-tein is a component of virions, rSV5 and rSV5DSH weregrown in MDBK cells, purified on sucrose gradients, andtheir polypeptide composition analyzed by SDS–PAGE.Polypeptides were immunoblotted using anti-SH IgGand, as a control for known virion components, wereimmunoblotted with sera specific for HN, P, and V pro-teins. As shown in Fig. 6, SH protein could be readilydetected in rSV5 virions. Attempts to show the presenceof SH in virions, using metabolic labeling of virions, ashad been used to show the presence of the M2 protein ininfluenza A virions (Zebedee and Lamb, 1988) were un-successful. However, SH protein only contains a single

TABLE 1

Mean Fluorescence Intensity (MFI) of F and HN Proteins in CV-1Cells Infected with rSV5 and rSV5DSH

Glycoprotein rSV5 MFI rSV5DSH MFI

F 706 6 60 601 6 10HN 171 6 15 146 6 5

Note. rSV5 and rSV5DSH virus-infected CV-1 cells at 18 h p.i. werewashed three times with phosphate-buffered saline deficient in cal-cium and magnesium and 0.02% sodium azide (PBS–FACS). The cellswere incubated with the F-specific mAb F1a (1:1000 dilution of ascitesfluid) (Randall et al., 1987) and rabbit anti-HN polyclonal antibody (1:300dilution) for 30 min on ice. The cells were then washed with PBS–FACSas described above and incubated with a goat anti-rabbit fluoresceinisothiocyanate-conjugated secondary antibody and a goat anti-mouseTexas-Red-conjugated secondary antibody for 30 min. The cells werewashed a further three times with PBS–FACS and fixed in 0.5% form-aldehyde. F- and HN-specific fluorescence on the aliquots were mea-sured on 10,000 cells using a FACSCalibur (Becton-Dickinson, SanJose, CA) flow cytometer. Values are means 6 SD.

34 HE ET AL.

methionine residue and no cysteine residues, which lim-its the usefulness of high specific activity radioactivemetabolic precursors. To determine the amount of the M2

protein in influenza A virions or the NB protein in influ-enza B virions, we have used a quantitative immunoblotmethod in which the signal from known amounts ofpurified virions was compared to that of known amountsof purified M2 or NB protein (Zebedee and Lamb, 1988;Brassard et al., 1996). However, for SH we have notsucceeded in generating a high-titer antisera useful foraffinity purification of the protein, and we have not suc-ceeded in generating an epitope-tagged version of SHthat is expressed at the cell surface (C. D. Ward and R. A.Lamb, unpublished data). Thus it is not possible to quan-tify the relative incorporation of SH into virions at present.

Examination of purified rSV5 and rSV5DSH virions byelectron microscopy indicated that for the .300 particlesof each virus examined, the morphologies of the virionswere indistinguishable (Fig. 7). Ideally it would useful toshow, in addition to immunoblotting, that SH protein iscontained in virions using immunoelectron microscopy.However, the only serum that is available to the SHprotein is the anti-SH IgG, which recognized a peptideencompassing the SH N-terminal cytoplasmic tail(Hiebert et al., 1985b, 1988). The SH ectodomain containsonly five residues, a size too small to permit generationof a specific serum. Thus examination of rSV5 andrSV5DSH by immunoelectron microscopy has to involvedetergent disruption of virions on the E.M. grid. Unfortu-nately, the experiment was not successful due to a lowsignal and nonspecific binding of the SH IgG to rSV5DSHvirions.

FIG. 7. Morphology of rSV5 and rSV5DSH. Purified rSV5 and rSV5DSH virions were absorbed onto electron microscopy grids and negatively stainedwith phosphotungstic acid. (A) rSV5, (B) rSV5DSH. Examination of .300 virions of rSV5 and rSV5DSH indicated no evident morphological differencesbetween the two virus populations. Bar, 50 mm.

FIG. 6. Western blot of purified rSV5 and rSV5DSH virions. rSV5 andrSV5DSH were grown in MDCK cells and purified through sucrosegradients. Virions were analyzed by SDS–PAGE on a 17.5% gel contain-ing 4 M urea, and polypeptides were transferred to a nitrocellulosemembrane. The blots were incubated with rabbit anti-SH IgG (left) ormouse anti-HN and anti-V/P sera (right) and appropriate HRP-conju-gated secondary antibodies. Bound HRP was detected by an enhancedchemiluminescent reaction.

35DELETION OF PARAMYXOVIRUS SV5 SH GENE

Growth rate of rSV5DSH

The growth rates of rSV5 and rSV5DSH were com-pared in a single-step growth curve experiment. MDBKcells were infected with the viruses at a m.o.i. of 10pfu/cell, the media were harvested at varying times, andvirus titers were measured by plaque assay. No differ-ence in plaque size between rSV5 and rSV5DSH wasobserved (Fig. 8A), and rSV5 and rSV5DSH showed verysimilar growth curves (Fig. 8B).

DISCUSSION

The data presented here indicate that it is possible torescue the paramyxovirus SV5 when it lacks its SH gene.Analysis of properties of the rSV5DSH virus indicatedthat deletion of the SH gene did not affect transcription ofthe F and HN genes, except that readthrough mRNAS(F–SH and SH–HN) could not be detected as would bepredicted for a SH gene deletion. The lack of expressionof the SH integral membrane protein did not alter theexpression levels of other viral proteins that were exam-ined (P, V, F, and HN) or the subcellular localization of theF and HN integral membrane proteins. Furthermore, lackof expression of SH did not affect the ability of the virusto cause fusion as measured by lipid mixing experiments

and content mixing experiments. The growth rate, infec-tivity, and plaque size of rSV5DSH indicated that theseproperties of the virus were directly comparable to thoseof rSV5. We also found that even though the SH proteinis a component of purified SV5 virions, the lack of SHfrom rSV5DSH does not alter the morphology of the virusparticles. Thus in tissue culture cells, the lack of the SV5SH protein does not confer a recognizable phenotype.However, the evolution of the GS and GE signals for SHindicates this is a specific transcription unit in the SV5genome and not a residual vestige of a pseudogene.Thus it seems quite possible that the absence of the SHprotein would confer a phenotype in an appropriate an-imal host. SV5 is naturally a pathogen of dogs, causingkennel cough, but SV5 has also been isolated frommonkeys, pigs, and humans (Choppin, 1964; Hull et al.,1956; Goswami et al., 1984; and Hans-Dieter Klenk, per-sonal communication). However, unfortunately at presenta small animal model that is fully permissive for the invivo study of pathogenesis is not available and the use oflarge animals is not fiscally feasible.

Among the Paramyxovirinae, only SV5 and mumpsvirus have SH genes (Hiebert et al., 1985a,b, 1988;Elango et al., 1989; Takeuchi et al., 1991, 1996; Afzal et al.,1993; Yeo et al., 1993; Kunkel et al., 1994, 1995), and theclosely related Rubulaviruses, hPIV2 (Kawano et al.,1990), hPIV4 (Bando et al., 1990), and simian virus 41(SV41) (Tsurudome et al., 1991) lack such a gene. Evenbetween SV5 and mumps virus there is a difference inthe SH proteins: although both SH proteins are smallintegral membrane proteins (44 and 57 amino, respec-tively), the available data suggest that the SV5 proteinhas its N-terminal domain in the cytoplasm (Hiebert etal., 1988), whereas the mumps virus SH protein has its Cterminus in the cytoplasm (Takeuchi et al., 1996). Further-more, with the Enders strain of mumps virus, a pointmutation in the F-gene polyadenylation site leads to theexclusion of monocistronic SH mRNA synthesis and pro-duction of only F–SH and F–SH–HN polycistronic mRNAs(Takeuchi et al., 1988), and there is no detectable expres-sion of the SH protein (Takeuchi et al., 1996), suggestingthat the SH gene may not be essential in tissue culturefor the replication of the Enders strain of mumps virus.However, it cannot be excluded that for the Enders strainof mumps virus undetectable amounts of essential SHmRNA and protein are synthesized.

The RS virus SH gene (originally named 1A) encodesa glycosylated integral membrane protein (Olmsted andCollins, 1989) that is often thought to be a counterpart ofthe SV5 SH protein (reviewed in Collins et al., 1996). Inearlier reports, it was found that expression of SH wasrequired for efficient RS virus-mediated fusion when vi-rus proteins were expressed from cDNAs using the vac-cinia virus expression system (Heminway et al. 1994).However, by using reverse genetics it was found possi-ble to delete the SH gene and recover a viable virus

FIG. 8. Single-step growth curve of rSV5 and rSV5DSH. MDCK cellswere infected with rSV5 and rSV5DSH at a m.o.i. of 10 pfu/cell andmedia harvested at 6-h intervals up to 36 h p.i. The virus titers weredetermined by plaque assay on BHK 21-F cells. (A) Plaques of rSV5 andrSV5DSH stained with Giemsa. (B). Growth curves of rSV5 andrSV5DSH.

36 HE ET AL.

(RSVDSH), and by a nonquantitative syncytium assay itwas found to cause fusion as efficiently as the parentalvirus (Bukreyev et al., 1997). Interestingly, the RSVDSHvirus showed improved growth in some tissue culturecells as compared to wild-type virus; however, theRSVDSH virus was attenuated in the upper but not thelower respiratory tract of mice (Bukreyev et al., 1997).Furthermore a cold-passaged strain of RS virus, B1 cp-52/2B5 has been found to contain a deletion of most ofthe SH and G gene sequences, yet this virus replicatesquite efficiently in tissue culture cells although it is highlyattenuated in RS virus seronegative infants and children(Karron et al., 1997).

Reverse genetics experiments using infectious cDNAclones of several Mononegavirale suggest that theseviruses contain dispensable accessory proteins that arenot required for replication in tissue culture. Neither theC protein of vesicular stomatitis virus (Kretzschmar et al.,1996) nor the similar in name (but not necessarily infunction) C protein of measles virus (Schneider et al.,1997) are required for replication in tissue culture but theeffect of these gene deletions in a suitable animal hosthas not been tested to date. Recombinant Sendai virusand measles virus that are unable to synthesize the Vprotein have also been recovered, indicating the V pro-tein is not essential for replication in tissue culture(Delenda et al., 1997; Kato et al., 1997a; Schneider et al.,1997). However, the Sendai virus V protein was found tobe essential for pathogenicity in the natural host, themouse (Kato et al., 1997a,b). Thus as the accessoryproteins of the Mononegavirale have been maintained inall natural viral isolates, it seems likely that they will havea role in determining viral pathogenesis in their respec-tive host organisms even though an appropriate hostanimal model system is not currently available for SV5 ormeasles virus.

MATERIALS AND METHODS

Plasmids

All molecular cloning procedures were carried outaccording to standard procedures (Ausubel et al., 1994).To generate pSV5DSH (pBH324), a DNA fragment(nucleotides 6303–6536, GenBank Accession No.AF052755) that covers the SH gene was deleted from theinfectious SV5 cDNA pSV5 (pBH276) (He et al., 1997). Thenucleotide sequence of all plasmids used is available onrequest together with details on the construction of theintermediate plasmids used to generate plasmids pSV5and pSV5DSH. The genome length of pSV5DSH wasengineered to conform to the rule of six (Calain andRoux, 1993) such that the total nucleotide number wasdivisible by six. The nucleotide sequence of the regioncovering the SH gene deletion was confirmed by dideoxychain terminating sequencing. Plasmids pUC19NP3A,pGEM2-P, and pGEM3-L are those plasmids described

previously (Thomas et al., 1988; Parks et al., 1992; Mur-phy and Parks, 1997). Plasmids containing the HN, F, andSH genes that were used for generating 32P-labeledprobes have been described (Paterson et al., 1984;Hiebert et al., 1985b; He et al., 1997). pYZ3 contains agene encoding bacteriophage T7 RNA polymerase undercontrol of the SV40 late region promoter and has beendescribed previously (Zhou et al., 1990). pBH82, whichcontains a gene encoding CAT under the control of a T7RNA polymerase promoter, control was described previ-ously (He et al., 1995).

Cells and viruses

Monolayer cultures of A549, MDBK, and CV-1 cellswere maintained in DMEM and 10% fetal-calf serum asdescribed previously (He et al., 1997; Paterson et al.,1984). Chick embryo fibroblasts (CEF) were grown asdescribed previously (Lamb and Choppin, 1976). BHK-21F cells were grown in DMEM with 10% fetal-calf serumand 10% tryptose phosphate broth.

Modified vaccinia virus Ankara (MVA) expressing bac-teriophage T7 RNA polymerase (Sutter et al., 1995; Wyattet al., 1995) was grown in CEF cells and was kindlyprovided by Dr. Bernard Moss, National Institutes ofHealth, Bethesda, MD. SV5 was grown in MDBK cellswith DMEM containing 2% fetal-calf serum as describedpreviously (Paterson and Lamb, 1993).

For single-step growth curves, MDBK cells in 3.5-cm-diameter dishes were infected with rSV5 or rSV5DSH ata m.o.i. of 10 pfu/cell. Media was collected at 0, 6, 12, 18,24, 30, and 36 h p.i. Viral titers were determined byplaque assay on BHK-21F cells as described (Patersonand Lamb, 1993).

Transfection of cDNAs encoding virus genomes andrecovery of rSV5 and rSV5DSH

A549 cells in a 3.5-cm-diameter plate were infected at95% confluence with MVA at an m.o.i. of 3 pfu/cell in PBScontaining 1% bovine serum albumin for 1 h, and then theplasmids pSV5, or pSV5DSH and those encoding NP, P,and L were transfected into cells with the cationic lipidLipofectin (Gibco-BRL, Rockville, MD). The amounts ofplasmids were as follows: 1.5 mg pSV5, 1.2 mg pUC19NP3A, 0.15 mg pGEM 2-P, and 1.5 mg pGEM 3-L. Infected/transfected cultures were incubated for 24 h, and thenthe transfection media was replaced with DMEM con-taining 2% fetal-calf serum and incubated for a further48 h at 37°C. The media were harvested, cell debrispelleted by low speed centrifugation (1250 x g, 5 min),and the supernatant filtered through a 0.45-mm filter toremove MVA. This virus stock was designated Pl andwas used to generate further SV5 stocks in CV-1 cells orMDBK cells. The virus was then clonally purified byplaque assay.

37DELETION OF PARAMYXOVIRUS SV5 SH GENE

Reverse transcriptase–PCR amplification

Total RNA from rSV5- or rSV5DSH-infected CV-1 cells(6-cm-diameter plates) was purified using an RNeasy kit(Qiagen, Chatsworth, CA) according to the manufacturer’sprotocol. RNAs were dissolved in H2O in a final volume of50 ml, and 19 ml was used in a 40-ml reverse transcriptase(RT) reaction containing 20 ng of oligonucleotide primerBH191 (sequence: 59-TATTGACCATTGTCGTTGCTAATCGA-AA-39) that annealed to the vRNA(2) strand of SV5. Analiquot of the cDNA was then amplified in a PCR reactionusing oligonucleotides BH191 and BH194 (sequence: 59-TCGAAATAATACTCGGCAAGTGGCC-39), which anneals tothe anti-genome sense RNA of SV5 (see Fig. 1). PCR wascarried out at 94°C for 1 min, 55°C for 1 min, and 72°C for1 min for 40 cycles. The PCR products were analyzed on a1% agarose gel and purified for sequencing using an ABI310 sequencer (Perkin-Elmer/Applied Biosystems, FosterCity, CA).

Northern blot analysis of mRNAs

Poly(A)-containing RNA from rSV5-infected CV-1cells (10-cm-diameter dish) was purified as described(Paterson et al., 1984) and separated by electrophore-sis on a 1.5% agarose gel containing 2.2 M formalde-hyde as described previously (Paterson et al. 1984).RNAs were transferred to a nitrocellulose membraneand crosslinked to the membrane by using UV irradi-ation (UV Stratalinker 2400, Stratagene, La Jolla, CA).a-[32P]-dCTP-labeled F, HN, or SH cDNA probes weregenerated using a Ready-to-Go random priming kit(Pharmacia Biotech, Piscataway, NJ). The membranewas hybridized with the probes and then washed un-der increasing stringency conditions (Paterson et al.,1984). The bound probes were detected by autoradiog-raphy.

Immunoprecipitation of polypeptides

CV-1 cells in 6-cm-diameter plates were infected withwild-type SV5, rSV5, or rSV5DSH at a m.o.i. of 3 pfu/celland at 22 h p.i., cultures were labeled with 100 mCi/ml[35S]Promix label (Amersham Life Science Products, Ar-lington Heights, IL) for 2 h. The cells were lysed in RIPAbuffer, and aliquots were immunoprecipitated using mAbHN4b specific for HN (Randall et al., 1987; Ng et al.,1989), a-F2 synthetic peptide rabbit sera (Horvath andLamb, 1992), mAb 161 specific for P protein (Thomas etal., 1988), a-V synthetic peptide rabbit sera (Paterson etal., 1995), and polyclonal rabbit serum raised to disruptedSV5 virions or rabbit anti-SH IgG (Hiebert et al., 1985b),as described previously (Paterson and Lamb, 1993).Polypeptides were analyzed by SDS–PAGE using 10 or15% polyacrylamide gels, except for SH protein, whichwas resolved on 17.5% polyacrylamide gels containing 4M urea. Radioactivity was analyzed using a Fuji BioIm-

ager 1000 and MacBas software (Fuji Medical System,Stamford, CT).

Fluorescent microscopy

CV-1 cells were grown on glass coverslips and in-fected with rSV5 or rSV5DSH. At 24 h p.i. cells werewashed with PBS and then fixed in 1% formaldehyde for15 min at room temperature. In all further steps, 0.1%Saponin was included in the PBS to permeabilize cells.The cells were washed with PBS/Saponin solution andincubated for 30 min in a 1:500 dilution of ascites fluid tomAb F1a specific for SV5 F protein, mAb HN1b specificfor the SV5 HN protein, and mAb Pk specific for the SV5P and V proteins or rabbit a-SH IgG. Cells were washedextensively in PBS/Saponin and the appropriate second-ary antibody [FITC-labeled goat anti-mouse IgG or Tex-as-Red-labeled goat anti-rabbit IgG (Jackson Laboratory,Bar Harbor, Maine)] were added to the cells for 30 min,and the cells washed in PBS/Saponin. Fluorescence wasexamined using a Nikon FXA fluorescent microscope(Nikon Corp., Tokyo, Japan) and appropriate filter sets.

Flow cytometry

rSV5 and rSV5DSH virus-infected CV-1 cells at 18 h p.i.were washed three times with PBS deficient in calciumand magnesium and containing 0.02% sodium azide(PBS–FACS). The cells were incubated for 30 min on icewith mAb F1a (1:1000 dilution of ascites fluid) and rabbita-HN polyclonal antibody (1:300 dilution). The cells werethen washed with PBS–FACS as described above andincubated with FITC-conjugated goat anti-rabbit IgG andTexas-Red-conjugated goat anti-mouse IgG for 30 min.The cells were washed a further three times with PBS–FACS and fixed in 0.5% formaldehyde. F- and HN-specificfluorescence was measured on 10,000 cells using aFACSCalibur flow cytometer (Becton-Dickinson, SanJose, CA).

Cell fusion assays

Lipid mixing fusion assay. The lipid mixing fusion as-say using octadecyl rhodamine (R18) was performedessentially as described previously (Morris et al., 1989).Human red blood cells were labeled with R18 as de-scribed (Bagai and Lamb, 1995). CV-1 cells on coverslipsin 6-cm-diameter plates were infected with rSV5 andrSV5DSH at a m.o.i. of 3 pfu/cell, and at 18 h p.i. the cellswere washed three times with PBS and incubated for 1 hat 37°C with 1 ml neuraminidase (50 mU/ml, SigmaChemical Co., St. Louis, MO). The cells were washedthree times with ice cold PBS, and 3 ml of 0.1% R18-labeled RBCs was added to the cells and incubated onice for 30 min with gentle shaking at 10-min intervals.The cells were then washed six times to remove un-bound RBCs. To activate fusion, the cells were incubatedat 37°C for different lengths of time. R18 fluorescence

38 HE ET AL.

was observed and recorded using a confocal micro-scope (Zeiss LSM410, Zeiss Inc, Thornwood, NY).

Cytoplasmic content mixing. BHK-21F cells in six-wellplates, at approximately 50% confluence were infectedwith rSV5 or rSV5DSH for 1 h and then transfected usingLipofectAmine (Gibco-BRL) with 1 mg of pYZ3 [whichcontains the gene encoding T7 RNA polymerase underthe control of the late region SV40 promoter (Zhou et al.1990)]. Concomitantly, BHK-21F cells in 6-cm-diameterplates were transfected with 2 mg of pBH82, which con-tains a gene encoding CAT under the control of a T7 RNApolymerase promoter and a 59 internal ribosomal entrysite from encephalomyocarditis virus (EMCV-IRES) (He etal., 1995). The cells were incubated in the transfectionmixture for 8–10 h and then trypsinized and overlayedonto virus-infected, pYZ3-transfected BHK-21F cells. Af-ter mixing of the two cell populations, the cells werecollected at different time points and CAT activity as-sayed as described previously (He et al., 1995).

Electron microscopy

rSV5 and rSV5DSH were grown in MDCK cells andpurified on sucrose gradients as described (Patersonand Lamb, 1993). For electron microscopy, purified viri-ons were absorbed onto parlodion-coated grids for 30 s,after which remaining virus suspension was washed offwith PBS as described previously (Jin et al., 1997). Gridswere stained with 2% phosphotungstic acid, (pH 6.6). Athin layer of carbon was evaporated over the grids priorto viewing in the electron microscope (Joel 120CX, Tokyo,Japan).

Immunoblotting

Purified rSV5 and rSV5DSH virus polypeptides wereanalyzed by SDS–PAGE on a 17.5% polyacrylamide gelcontaining 4 M urea. The polypeptides were transferredto PVDF membranes by using a semidry transfer (Trans-blot) apparatus (Bio-Rad Inc., Richmond, CA) and mem-branes immunoblotted as described previously (Bur-nette, 1981). Immunoblotting was performed by using1:1000 dilutions of the a-SH IgG, a-HN mAb HN5a, anda-V/P mAb Pk. HRP-conjugated antibodies were used assecondary antibodies. The bound antibodies were de-tected by enhanced-chemiluminescence with the Super-Signal Substrate kit (Pierce Inc., Rockford, IL) accordingto the manufacturer’s instructions.

ACKNOWLEDGMENTS

We thank Helen Nestoras for excellent technical assistance. Plas-mids pYZ3 and pBH82 were the kind gift of Dr. W. T. McAllister. Thiswork was supported by Research Grant AI-23173 from the NationalInstitute of Allergy and Infectious Disease. B.H. is an Associate andR.A.L. is an Investigator of the Howard Hughes Medical Institute.

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